The overall goal of this proposal is to develop a new cost-effective method for the production of high-purity polyclonal antibodies. Polyclonal antibodies are important reagents used worldwide for antigen detection and purification in basic research as well as the detection of disease states. Increased availability of quality anti-peptide polyclonal antibodies would greatly benefit proteomic research. Unfortunately the production of polyclonal antibodies to peptide antigens currently requires the chemical synthesis and purification of peptide antigens, which is expensive and time-consuming. The proposed research will result in the development of new methods to produce purified polyclonal antibodies. These methods will allow the rapid production of recombinant peptide antigen/immunogen conjugates while eliminating both the need for chemical synthesis of peptide antigens as well as the chemical conjugation of peptide haptens to carrier immunogens such as KLH. The new production method will be amenable to high throughput production of high quality antibodies at a lower cost. Specific aims for Phase I: (1) Screen a series of proteins to identify new highly immunogenic proteins which can be recombinantly expressed in bacteria. (2) Develop a plasmid vector to allow the rapid cloning, expression and purification of peptide antigens fused to carrier immunogens identified in the screen in Aim 1. (3) Produce polyclonal antibodies to several peptides produced by immunizing rabbits with recombinant peptide-immunogen fusion proteins. Characterize the antibodies to identify the best overall carrier immunogen. In Phase I we will identify new immunogens which can be recombinantly-expressed in bacteria and then create an expression vector to allow the rapid cloning and expression of peptide antigens fused to the carrier immunogens. We will use protein-engineering techniques to improve the immunogenicity of the carrier even further. We will clone several peptide antigen sequences into the vector and express and purify the peptide-immunogen fusions from bacteria. We will then produce polyclonal antibodies in rabbits using the purified peptide fusion proteins. We will purify the antigen-specific antibodies from the serum using a two-step affinity-based procedure. Finally, we will evaluate the performance of each of the antibodies side-by-side and select the best performing carrier for further commercial development in Phase II. In Phase II, we will scale-up the most efficient system and broaden its utility for the production of a variety of diverse peptide antigens. We will also develop molecular biology techniques to incorporate modified amino acids (such as phosphoamino acids) into the peptide antigens expressed in bacteria. Anti-peptide polyclonal antibodies are important reagents for the detection and characterization of proteins in cells and other complex biological systems. The exquisite specificity of anti-peptide antibodies has been used to develop sensitive assays to examine characteristic changes in the proteome associated with diseased states. The ability of researchers to develop these assays is currently limited by the availability of high-quality antibodies. In Phase I we will develop a novel, commercially-viable method for high-throughput production of anti-peptide polyclonal antibodies. In Phase II, we will scale-up the most efficient system and broaden its utility for the production of a variety of diverse peptide antigens. We will also develop molecular biology techniques to incorporate modified amino acids (such as phosphoamino acids) into the peptide antigens expressed in bacteria. Our work will greatly improve the availability of low cost, high quality antibodies to the worldwide research community. [unreadable] [unreadable] [unreadable]